The long-term goal of this project is to characterize the mechanisms that distinguish germ cells from somatic cells, a fundamental problem in developmental biology. In C. elegans, soma- germline asymmetries are established in the zygote before the first division, through the asymmetric partitioning of proteins and protein/RNA complexes in the cytoplasm. Cytoplasmic partitioning is regulated by conserved polarity regulators (PAR proteins), which localize asymmetrically in the zygote cortex. The goal of this proposal is to uncover how PAR activity at the cortex regulates polarity in the cytoplasm. Specific Aim I (SA1) will focus on the mechanisms that segregate the RNA-binding protein MEX-5 to the anterior cytoplasm. Preliminary data suggest that MEX-5 asymmetry depends on phosphorylation by PAR-1, a kinase on the posterior cortex, which increases MEX-5 diffusion locally in the posterior cytoplasm. SA2 will focus on the germline determinant PIE-1, which segregate to the posterior, opposite MEX-5 and in a steeper gradient. We will investigate how PIE-1 integrates both cortical and cytoplasmic cues to form a distinct gradient. Finally, SA3 will focus on the mechanisms that localize P granules to the posterior. P granules are evolutionarily conserved RNA-protein complexes specific to the germline. We have discovered a new P granule component PPTR-1 for P granule integrity during mitosis; our initial findings suggest that the germline specificity of P granules depends on regulated assembly and does not require cytoplasmic partitioning. We will use a combination of biochemical, genetic, and live microscopy approaches to test each of these hypotheses, and identify the genes and biochemical interactions involved. Unlike other well-studied embryonic polarity models, our system does not rely on pre-localized RNAs to generate asymmetry, and thus gives us the unique opportunity to explore the mechanisms that directly segregate proteins in the cytoplasm.